Structural and biochemical insights into the mechanism of the Gabija bacterial immunity system

The Gabija system is a newly discovered bacterial immune system that consists of GajA and GajB. Here we report the cryo-EM structure of the Gabija complex from Bacillus cereus VD045 at 3.6 Å, which provides the direct evidence of interactions between GajA and GajB. The Gabija complex is an octameric ring structure with four GajA and four GajB. GajA is an OLD nucleases family protein, while GajB belongs to the SF1 helicases. The Gabija complex has sequence-specific DNA nuclease activity and prefers circular rather than linear DNA as substrate, its activity is more sensitive to concentrations change of nucleotides compared to GajA alone. Our data suggest a mechanism of Gabija immunity: the nuclease activity of Gabija complex is inhibited under physiological conditions, while it is activated by depletion of NTP and dNTP upon the replication and transcription of invading phages and cleave the circular DNA to prevent phage DNA replication.

Gabija is a recently idenfified bacterial immune anfiviral defense system that consists of GajA and GajB proteins.The exact mechanism of Gabija immune funcfion is currently unknown however it is evident from prior published data that both GajA and GajB are required.In this manuscript, Huo and colleagues provide new structural and funcfional data to support a mechanism of GajA nuclease acfivity being regulated through direct protein-protein interacfion with GajB and build on prior work to reveal a marked dependence on intracellular nucleofide concentrafion.The authors provide the first reported cryo-EM structure of the complex formed between GajA and GajB revealing a 4:4 rafio of subunits.Evidence is also provided that the Gabija complex is potently acfivated (or rather disinhibited) to cleave circular DNA by low ATP concentrafion and inhibited at higher levels.There is a high level of interest in the field for structural and biochemical characterizafion of this newly defined defense system and as such these data are valuable.The manuscript in its current form does provide an intriguing model for how free-nucleofide deplefion during phage replicafion could lead to acfivafion of the Gabija system.However, the data and figures are presented in a way that is inconsistent with the standards and expectafions of the field and separately, some of the main hypotheses generated in this manuscript will likely need to be tested by experiments with phage.Outlined below are suggesfions and comments that may aid in producfive revision of the manuscript before resubmission for peer-review.

Concerns:
• Abstract line 1: The word "predicted" here does not apply any longer as published evidence for immune acfivity of this system is cited by the authors.
• Introducfion, second paragraph: "Sorek et al. predicted computafionally and validated experimentally 10 previously unknown bacterial immune defense systems3,4."The reference #3 to the 2018 Sorek et al. manuscript (should read Doron et al.) is correct but reference #4 has no relevance to this manuscript nor to the sentence for which the citafion is aftached and should therefore be removed.
• References to many relevant and more recent work in the field of prokaryofic innate immune systems is lacking in this manuscript.In fact, only 8 references to non-software/cryo-EM methods papers are cited which feels very limited.It should be menfioned in greater detail that Gabija is one of now 100s of newly idenfified immune defense mechanisms in prokaryotes and appropriate references cited.
• Introducfion: "We further proved that the Gabija complex has the sequence-specific DNA endonuclease acfivity and prefers supercoiled circular DNA as its substrate."The word "proved" is too strong/absolute.Certainly, evidence has been provided that suggests the Gabija complex has DNA sequence preference and prefers supercoiled circular DNA.However, this has not yet been shown in vivo.
• Generally, the color choices in the structure figures are inconsistent between panels and not beneficial to the reader.The abundance of colors used is overwhelming and contrast is not used appropriately.
• In Figure 1, labeling of the individual subunits A-G is less useful than highlighfing GajA vs GajB.Represenfing the hetero-octameric structure could be done more clearly by showing one heterodimer in colors and the other three in grey (for example).
• In Figure 4A, showing hydrogen atoms on all the lysine residues is not useful or accurate especially given the resolufion of the cryo-EM data and addifionally Figure 4B is somewhat redundant with 4A.The authors could choose to just show 4B alone as there is more informafion content.
• Page 4-further explanafion is needed for "ATPase domain of GajA adopts a producfive conformafion." • Page 7 and Figure 3-What is the RMSD value for the structural alignment of the structures (GajA and OLD)?
• Pages 8 and 11-The authors did not menfion how the mutants were created in the methods secfion.Addifionally, authors did not confirm/indicate if the mutants were properly folded.
• Extended Data Figures 4 and 6-The authors must show individual data points not just the average and error bars.The stafisfical analysis used to generate the error bars need to be explained in the figure legend (SEM?SD?) • Extended Data Figures 4 and 6-Why are these acfivity plots separated out into two separate figures?The GTPase level of GajB appears much weaker than the ATPase rate.Can the authors comment more on this subject?Possibly also tesfing GTPase acfivity for the GajA and GajAB complex?
• "The octameric ring formafion can be best described as three steps: protomer formafion of GajA-GajB heterodimer and two sequenfial dimerizafion."In this descripfion the authors make it appear that they know the order of steps leading to Gabija complex formafion.The language should be modified to reflect that this is hypothefical.
• What does "The cleavage acfivity of the Gabija complex on supercoiled plasmid is not affected at all" mean?Was this a comparison to the GajA protein alone?
• "This substrate selecfivity is more efficient in bacterial immune response…" needs to be explicitly tested/shown if this phrase is to remain in the manuscript.Indeed, the following sentence needs to be jusfified through experiment as well.
• In the discussion secfion-"Although GajB was predicted as a UvrD-like SF1 helicase" what is the intent behind use of the word "although" in this context?
• The descripfion of the model for how GajAB is acfivated by nucleofide deplefion during phage infecfion and its overall funcfion in immunity could benefit from a figure panel to help guide readers.
• As Alphafold2 models for GajA and GajB were docked into the maps that were obtained, it would also be useful to present/discuss how different the models were relafive to the final build.Were the conformafions differing significantly from what was predicted?It is menfioned that some "density" was missing such that the full structures were not able to be built into the map.It might help to show this analysis in an extended data figure.
• Page 5-define CTR and NTR -C-terminal region and N-terminal region.

Point-by-point Response to Reviewers' Comments
Reviewer #1 (Remarks to the Author): Structural and biochemical insights into the mechanism of the Gabija bacterial immunity system Huo et al.
I was asked to review the cryo-EM technical aspects of this manuscript, something I find myself unable to do due to the authors' refusal to abide by the data transparency standards in the field.
This began with the initial submission.Surprisingly, a manuscript whose main finding was a cryo-EM structure of the Gabija complex was virtually devoid of cryo-EM data.Other than an incomplete cryo-EM table, the manuscript had no figures, either main or supplementary, showing their cryo-EM map, data processing, model fit, etc.I wrote to the editor to point this out and to say I would look at the manuscript once that data was available.
A week later, the editor let me know that a supplementary figure on cryo-EM data processing, and a PDB Validation Report had now been submitted.That turned out not to be entirely true.While there was a supplementary figure outlining the cryo-EM data processing, it is still inadequate.The FSC curve does not show all the standard curves, and there is no indication (visual or quantitative) of how well the model fits the map.Worse, the PDB validation report is clearly labeled as "NOT for manuscript review", because the authors had deposited neither their PDB model, nor the raw full and half cryo-EM maps needed for resolution estimation and map-to-model fit calculations.As clearly stated in the report, it was generated by the authors using the validation server.I wrote to the editor again, letting her know I would not review the paper until the maps and models had been deposited in the EMDB and PDB, and a new validation report, fit for review, was generated.
A few days later, the editor sent me a link to a folder with cryo-EM data: filtered map, raw half maps, and model.However, as far as I can tell the authors have not yet deposited these data in the PDB and EMDB, and thus still lack a PDB Validation Report.Furthermore, even the data provided in that folder appears incorrect.The unfiltered half maps look strongly filtered and are visually clearly of much lower resolution than the final filtered map.This makes no sense as it is the opposite of what one would expect: the half maps should be noisier (i.e.look more "detailed") than the filtered map.
The authors must deposit their data and include PDB and EMDB entry numbers in their manuscript, along with a PDB Validation Report fit for review.More cryo-EM data is needed in the manuscript as well to let readers judge the structure without having to obtain maps and analyze them themselves.If the authors refuse to abide by current standards in scientific publishing, I am unwilling to waste my time reviewing this manuscript.We are no longer in the 1990s, when structures were routinely published with no data made available.

Response:
We are very grateful for your objective and professional comments on the cryo-EM technical aspects of the manuscript.We deeply regret the unprofessional manner in which we handled the structural data in the previous version of our manuscript, causing confusion for reviewers and inconvenience to the editor.We genuinely apologize for any frustration this may have caused.

Initial Submission:
We acknowledge that our initial submission lacked essential cryo-EM data, which was an oversight on our part.We apologize for any inconvenience this may have caused.It is important to clarify that we have never refused to adhere to current standards in scientific publishing.Upon receiving the request from the editor, we promptly took the necessary steps to rectify the situation.Specifically, we deposited the cryo-EM density map and the related model into the EMDB and PDB databases.Additionally, we provided the cryo-EM data as a supplementary file along with the cryo-EM density map and related model.We understand your concern regarding the delay in providing a formal validation report for manuscript review.The time required for its generation was beyond our control and currently more than 7-10 days (26 days in our case).We have submitted the formal validation report after the initial submission.

Supplementary Data:
We acknowledge the insufficiency of the supplementary figure on cryo-EM data processing.Your comments regarding the FSC curve and the absence of quantitative information on model fit are duly noted.We are committed to providing a comprehensive and transparent account of our cryo-EM data and will ensure that these aspects are properly represented in our revised manuscript.

Cryo-EM Data Folder:
We appreciate the feedback regarding the cryo-EM data provided in the folder.We have carefully reviewed the data to ensure the accuracy and confirmed that the halfmaps provided earlier were directly generated by the software Relion without any other processing.Additionally, we will make sure to deposit these data in the PDB and EMDB as you have rightly suggested.
Incorporating More Cryo-EM Data: We agree with your suggestion to include more cryo-EM data in the manuscript to facilitate readers' understanding and evaluation of the structure.We are committed to providing a more comprehensive representation of our cryo-EM findings, allowing for a thorough assessment without requiring readers to obtain and analyze maps independently.
Specifically, we added more information in Extended Data Fig. 3, Extended Data Fig. 4, Extended Data Fig. 12 and Extended  I am safisfied with the changes the authors have made to the manuscript to include a reasonable amount of cryo-EM data for readers to judge the quality of the structures.Maps and models have been deposited and PDB Validafion reports for reviewers are now included in the re-submission.
Reviewer #2 (Remarks to the Author): In this revised manuscript, the authors have responded to my queries and provided further data on nonhydrolysable analogues of ATP.
Regarding point 2, the extended recognifion sequence, the authors' response doesn't really add much to the analysis.Thinking about the recognifion site, it is possible that the palindromic T/A sequences allow formafion of a small hairpin in DNA.This would be preferenfially formed in supercoiled DNA that is negafively supercoiled, which would fit the data presented in the paper.There are ways to test this, for example by making a fixed four-way juncfion in linear DNA using the sequence.However, given this is review stage 2 and there are other papers in press it might be best just to menfion the possibility in the discussion.
Reviewer #3 (Remarks to the Author): This reviewer is mostly safisfied by the substanfial new experimental data and analysis provided by the authors in this revised version of the manuscript.Addifional evidence for the folding and enzymafic acfivity of Gabija is now included that strengthens the original draft.
The plaque assay presented in Extended Data Figure 2 appears possibly overloaded and 7-8 logs of protecfion is almost unbelievable.Trusfing that all sample lanes were loaded the same, the phenotype is remarkable.A minor concern is that there is limited ufility of the color scheme used in Extended Data Figure 8.Consider another version that may rely less on readers ability to disfinguish between closely related or otherwise complementary colors.
Data Table 1.To ensure transparency and accessibility, we have deposited the cryo-EM density map and related model coordinate in the databases EMDB and PDB with accession codes EMD-35977 and 8J4T, respectively.